In the intestinal lumen, protein hydrolysate increases the transcription and release of cholecystokinin (CCK) from enteroendocrine cells of the duodenal-jejunal mucosa. Our recent discovery that a G protein-coupled receptor, GPR93, is activated by dietary protein hydrolysate causing induced intracellular calcium-mediated signaling events in intestinal epithelial cells raises a possibility that GPR93 might be involved in the protein hydrolysate induction of CCK expression and/or secretion. Using the enteroendocrine STC-1 cells as a model, the present study demonstrates that increasing expression of GPR93 amplifies the peptone induction of endogenous CCK mRNA levels. A similar increase in CCK transcription, indicated by the luciferase reporter activity driven by an 820-bp CCK promoter, is also observed in response to peptone at a dose as little as 6.25 mg/ml, but not to lysophosphatidic acid (LPA), an agonist of GPR93. We discovered that the upregulation of CCK transcription involves ERK1/2, PKA, and calmodulin-dependent protein kinase-mediated pathways. Additionally, GPR93 activation by peptone induces a response in CCK release at 15 min, which continues over a 2-h period. The cAMP level in STC-1 cells overexpressing GPR93 is induced at a greater extent by peptone than by LPA, suggesting a possible explanation of the different effects of peptone and LPA on CCK transcription and secretion. Our data indicate that GPR93 can contribute to the observed induction of CCK expression and secretion by peptone and provide evidence that G protein-coupled receptors can transduce dietary luminal signals.
- lysophosphatidic acid
enteroendocrine cells have a characteristic flask-shaped morphology with the apical plasmalemma that is in contact with the luminal contents and the basolateral surface that is in contact with the vasculature of the lamina propria, neural cells, and distant enterocytes through cytoplasmic projections. In direct response to nutrient and nonnutrient stimuli, gastrointestinal endocrine cells secrete bioactive peptides from their basolateral membrane surface that can act as endocrine, autocrine, paracrine, and neurocrine agents. Therefore, nutrient absorption and assimilation as well as metabolic and behavioral responses could be modulated as a result of luminal stimulation of enteroendocrine cells.
Luminal products of digestion can act not only as secretagogues of bioactive peptides but also as transcriptional activators of those peptide genes. For example, the secretion and transcription of gastrin in the stomach are elevated in response to luminal protein (4). Luminal protein hydrolysate (peptone) increases the secretion of glucagon-like peptide-1 (GLP-1) and the transcription of proglucagon in the intestine (19), as well as inducing cholecystokinin (CCK) secretion and gene transcription (33, 39).
Although the physiological role of G protein-coupled receptors (GPCRs) as transducers of luminal sensing in enteroendocrine cells remains unclear, there is increasing evidence of a possible role for GPCRs as molecular sensors on the surface of enteroendocrine cells that are responsive to luminal contents. G proteins, such as gustducin, that are coupled to oral taste receptors are also found in the intestine (7, 51, 52). The calcium-sensing receptor is also expressed in the gastrin-secreting antral cells and is responsive to aromatic amino acids (11, 40). GPR40, which can be found in the intestine (5), is activated by free fatty acids (FFA) of specific chain length and is sensitive to calcium channel blockers, in a manner that parallels FFA-induced CCK release in enteroendocrine STC-1 cells (17, 37). This suggests that FFA-induced CCK release may be mediated by GPR40 or a similar GPCR.
Protein hydrolysate stimulates CCK secretion from STC-1 cells (13); however, the mechanism of that induction is unknown. We recently reported that protein hydrolysate, a physiological model of dietary protein found in the lumen after a meal (14, 15), directly activated GPR93 in enterocytes, resulting in the mobilization of intracellular calcium concentration ([Ca2+]i) and the activation of ERK1/2 through both Gαq- and Gαi-mediated pathways. These data suggested that GPR93 could be partly responsible for the induced ERK1/2 activation observed in the enterocytes leading to alterations in cell proliferation and differentiation (10). This also brought forward the possibility that GPR93 could be a nutrient-responsive GPCR capable of inducing CCK release through the elevation of [Ca2+]i in the CCK-releasing enteroendocrine I cells.
In the present study, we identify GPR93 in the CCK-secreting STC-1 cells and show that the exogenous expression of this receptor in these cells results in a dose-dependent increase in [Ca2+]i in response to protein hydrolysate. The activation of GPR93 by protein hydrolysate also increases CCK gene transcript level. Additionally, STC-1 cells overexpressing GPR93 display a significant increase in CCK transcription and release in response to protein hydrolysate. This study demonstrates for the first time that GPCRs, exemplified by GPR93, can be molecules that are important in the signaling mechanisms involved in the nutrient induction of the transcription and secretion of bioactive peptides, such as CCK, in enteroendocrine cells.
MATERIALS AND METHODS
STC-1 cells (passage 17) were kindly provided by Dr. Douglas Hanahan (University of California, San Francisco). Other reagents used in this study were purchased from Sigma Aldrich unless indicated differently.
Cell culture condition.
STC-1 cells (passage 25 to 35) were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) with 10% fetal bovine serum (FBS; Hyclone Laboratories), 2 mM l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin as additional supplements; at 37°C in 5% CO2/air. The Berkeley rat intestine epithelial 380 hybrid cells (hBRIE 380i, passage 12 to 18) were maintained as previously described (10).
The GPR93 and the mitochondria-targeted aequorin (mtAEQ) expression plasmid constructs were as described previously (10). The CCK promoter-linked luciferase reporter plasmid described by Bernard et al. (2) was constructed as follows: a rat genomic DNA fragment from −728 to +91 bases relative to the CCK gene transcription starting site was isolated by PCR using Pfu DNA polymerase (Stratagene). The forward primer, 5′-CCTCCTCGAGAAAGGAAGAACTCTAGAGGACGGGAAGATCATTGC-3′, and the reverse primer, 5′-CACCATGGCTGGCTTGGCGGTTTCCAACGGCTGCTGTC-3′, that were used to isolate the promoter fragment were designed to produce a PCR fragment with XhoI and NcoI sites on its 5′ and 3′ ends, respectively. The resulting PCR fragment was digested with XhoI and NcoI restriction enzymes and ligated to the corresponding sites of a luciferase reporter vector, pGL3-basic (Promega). All constructs were verified by DNA sequencing (DNA Sequencing Facility, University of California, Berkeley).
For the comparison of endogenous GPR93 expression in both hBRIE 380i cells and STC-1 cells, hBRIE 380i cells were plated in T25 flasks (2.5 × 105 cells/flask) in IMDM-10% BCS, and STC-1 cells were plated on 35-mm tissue culture dishes (1.6 × 105 cells/dish) in DMEM-10% FBS. The hBRIE 380i cells at preconfluency (proliferating stage), confluency, or 8 days postconfluency (differentiated stage) and the STC-1 cells at confluency were harvested by addition of 1 ml of TRIzol (Invitrogen)/flask or dish, after being rinsed three times with PBS. The total RNA from the TRIzol homogenate was isolated according to the manufacturer's protocol.
To determine the change of the endogenous CCK gene expression by GPR93, 3.2 × 105 STC-1 cells/dish were laid down on 35-mm tissue culture dishes, 24 h before transfection. Either GPR93 plasmid or the empty vector (pCI-neo; Promega) (0.6 μg/dish) was then transfected into the cells by using Superfect reagent (Qiagen) according to the manufacturer's protocol, and cells were allowed to recover for 24 h under the normal culture condition. Before the treatment, cells were rinsed three times with PBS and then serum starved in DMEM-0.1% fatty acid-free bovine serum albumin (FBSA) for 12 h. Cells were treated with various doses of LPA or peptone, as indicated in the figure, for an additional 6 h. At the end of the treatment, 1 ml of TRIzol/dish was added and RNA was isolated.
Reverse transcription was performed as previously described (31). The PCR primers for GPR93 were designed to match the GPR93 sequence of rat (accession number: XM_575667) and mouse (accession number: BC117528). The GPR93 forward primer sequence was 5′-GCTCTGCCTGGGCGTGTGGGCTCTCATCCTGC-3′ and the reverse primer sequence was 5′-GCGTCGGGCCTCGCCAGTGTCCAGAAGAC-3′. The sequence of the PCR primers for CCK (accession number: BC028487) analysis was forward primer 5′-AAAGCCATGAAGAGCGGCGTATGTCTGTGCGTG-3′ and reverse primer 5′-ATTAGAGGCGAGGGGTCGTATGTGTGGTTG-3′. The sequence of the PCR primers for glucose-dependent insulinotropic peptide (GIP) (accession number: BC104314) analysis was forward primer 5′-CAGAGGGGACTTTCATCAGTGATTAC-3′ and reverse primer 5′-GCCAGTAGCTCTTGAATCAGAAGGTC-3′. The PCR primers for the ribosomal 18S RNA and villin were described previously (31). Taq DNA polymerase (New England Biolabs) was used to PCR amplify cDNA fragments of CCK (429 bp), GIP (256 bp), villin (516 bp), and 18S (542 bp). The PCR parameters were 20 s at 94°C, 15 s at 55°C, and 30 s at 72°C, for 19–35 cycles. The amplified cDNA fragments were analyzed by agarose gel electrophoresis followed by densitometry. The level of 18S RNA expression in each sample was used to normalize the RNA content of samples. A PCR standard curve was used for the semiquantitative measurement of CCK level. The 429-bp cDNA fragment of CCK gene was purified and used as the standard. The purified fragment was serially diluted, PCR amplified with the same cycle parameters as used for the samples, and densitometrically quantitated to determine the appropriate concentration range for a standard curve for a given RT-PCR result. The band intensity of the CCK cDNA fragments from the samples was then compared with the standard curve, and the fold change was calculated by arbitrarily designating 1 as the value for no treatment. The identity of the PCR products was confirmed by DNA sequencing.
Aequorin-based [Ca2+]i mobilization assay.
The [Ca2+]i mobilization assay was performed as previously described (10). Briefly, cells were laid down 24 h before the transfection (2 × 106 cells/T25 flask). The mtAEQ expression vector (3 μg plasmid DNA/flask) was cotransfected with either the GPR93 expression plasmid or empty vector (3 μg DNA/flask) by using Superfect reagent according to the manufacturer's protocol. Twenty hours after transfection, cells were trypsinized and allowed to recover in DMEM-10% FBS with gentle rolling for 1 h, followed by loading with 5 μM coelenterazine-h (Promega) and 300 μM glutathione in DMEM with gentle agitation for an additional 1 h. Cells were then centrifuged at 100 g for 5 min at room temperature and resuspended in Hanks’ balanced salt solution (HBSS). A 100-μl aliquot (1 × 105 cells in HBSS) was assayed in a luminometer equipped with an injector (Turner BioSystem). Peptone or LPA at the concentrations indicated in the figure was injected into the cell suspension in a 100-μl aliquot at a 2 × concentration in PBS. A 100-μl aliquot of lysis buffer (300 mM CaCl2, 300 μM digitonin) was injected 40 s later to react with the remaining aequorin. The luminescence (as relative light units, RLU) was recorded continuously. Fractional RLU is defined as the increased RLU due to a stimulus normalized to the total RLU, i.e., the integrated RLU value for 30 s after injection of the stimulus plus the value for 20 s after the addition of the lysis buffer. All stimuli were dissolved in PBS (pH 7.4).
Luciferase reporter assay.
Cells were laid down on 24-well plates (8 × 104 cells/well), 24 h before transfection. The CCK promoter linked luciferase reporter plasmid (80 ng DNA/well) was cotransfected with the GPR93 expression plasmid or empty vector (80 pg DNA/well) by use of Superfect reagent according to the manufacturer's protocol and allowed to recover for 24 h. On the day of the experiment, cells were first washed three times with PBS and serum starved for 12 h in the presence of 0.1% FBSA and then treated with either 10 μM LPA or 6.25 to 25 mg/ml peptone in DMEM-0.1% FBSA for 6 h. At the end of the treatment, cells were lysed with the addition of passive lysis buffer (Promega) (40 μl/well). The reporter activity of each sample was determined according to the manufacturer's protocol using a luminometer and normalized to the total protein concentration determined by the Bradford method (Bio-Rad).
To determine the effect of pertussis toxin (PTX) on [Ca2+]i mobilization by GPR93 activation, cells were transfected with the mtAEQ and GPR93 plasmids as described under Aequorin-based [Ca2+]i mobilization assay and incubated in media containing 50, 100, or 200 ng/ml PTX for 5 h before the assay. To determine changes in luciferase reporter activity in the presence of intracellular calcium chelator or inhibitors of MEK1/2, calmodulin-dependent protein kinase (CaMK), or PKA, cells were preincubated with 20 μM BAPTA-AM; 15, 30, 50, or 80 μM PD98059; 2, 3, or 10 μM KN93; or 2, 5, or 10 μM H-89, for 2 h before the addition of LPA or peptone.
Measurement of the CCK release.
STC-1 cells were laid down on 12-well plates (1.6 × 105 cells/well) 24 h before the transfection. The cells were transfected with the GPR93 expression plasmid or empty vector plasmid (0.8 μg DNA/well) by using Superfect reagent according to the manufacturer's protocol and then allowed to recover for 48 h. On the day of the experiment, cells were first washed three times with PBS and incubated in HBSS-0.1% FBSA with various stimuli for various periods of time as indicated in ⇓⇓⇓⇓⇓Fig. 6 at 37°C. At the end of the experiment, the plates were placed on ice and the media were collected from each well and centrifuged at 300 g for 5 min at 4°C to remove residual cells. Subsequently, 250 μl of cold 0.1 N HCl was added to each well, and then cells were scraped from each well. The cell lysate from each sample was centrifuged at 16,000 g for 5 min at 4°C, and the supernatant was neutralized with 25 μl of 1 N NaOH before determination of CCK concentration. The CCK concentration in both cell lysate and media was determined by enzyme immunoassay using an enzyme immunoassay kit (Phoenix Pharmaceuticals). The concentration of CCK was normalized to the total protein content in each well.
The lowest detectable level of CCK under our experimental condition was 0.05 ng/ml. The interassay coefficient of variance at the lowest detectable level was 11.31%. Although the antibody used in enzyme immunoassay to determine the CCK level in STC-1 cells can cross-react with human gastrin, all immunoreactivity detected is assumed to be CCK since STC-1 cells do not synthesize a detectable level of gastrin (37).
Measurement of intracellular cAMP.
STC-1 cells were laid down on 24-well plates (8 × 104 cells/well) 24 h before the transfection. The cells were transfected with the GPR93 expression plasmid or empty vector plasmid (0.4 μg DNA/well) by using Superfect reagent according to the manufacturer's protocol and then allowed to recover for 48 h. On the day of the experiment, cells were first washed three times with PBS and incubated in HBSS-0.1% FBSA for 30 min, followed by an additional 30 min incubation in the presence of 1 mM of 3-isobutyl-1-methylxanthine. Cells were then treated with either 50 mg/ml peptone or 10 μM LPA for 7 min. The treatment was terminated by placing the cells on ice and rinsing three times with ice-cold PBS. After an addition of 100 μl/well of 0.1 M HCl, the cells were scraped on ice and the cytosolic fraction of each sample was obtained by centrifugation (10,000 g for 10 min at 4°C). The cAMP concentration was determined by an enzyme immunoassay method (Cayman Chemical). The interassay coefficient of variance was 20%, and the lowest detectable level of cAMP was 3 nM.
Data are expressed as means (SD). Statistical difference between multiple groups was determined by one-way ANOVA with Tukey's post hoc test performed by using SPSS version 11. Significance was accepted at P < 0.05.
Our recent discovery of GPR93 expression in the intestinal mucosa and its activation by peptone in enterocytes (10) led us to investigate whether GPR93 was also activated by peptone in CCK enteroendocrine-like cells. We used STC-1 cells that have been well studied as a model system for peptone-induced CCK secretion. To increase the confidence that what we examined could be physiologically relevant, it was necessary that GPR93 was endogenously expressed in STC-1 cells, allowing us to assume that the downstream effectors of GPR93 activation could also be endogenously expressed.
We compared the endogenous GPR93 expression in STC-1 and enterocyte hBRIE 380i cells by RT-PCR analysis. STC-1 cells expressed GPR93 at a level comparable to confluent hBRIE 380i cells (Fig. 1).
There is a significant body of literature describing multiple effects of peptone in STC-1 cells. Considering the possibility that more than one factor might mediate a particular effect, we chose to elevate the level of GPR93 expression, for this study, by transient transfection (Fig. 2, inset).
Peptone induces an increase in [Ca2+]i in STC-1 cells overexpressing GPR93.
GPR93 activation by peptone in both CHO and hBRIE 380i cells induces [Ca2+]i flux, as determined using mtAEQ as an indicator of [Ca2+]i mobilization (10). Similar to those results, peptone as well as LPA, a potent agonist of GPR93, enhanced the increase in [Ca2+]i in a dose-responsive fashion (Fig. 2). However, the magnitude of the enhancement of [Ca2+]i increase in STC-1 cells was lower than what was observed in hBRIE 380i cells (10). An induction by LPA in STC-1 cells, similar to what was observed in hBRIE 380i cells, required elevated LPA concentrations. A transient [Ca2+]i increase that could be observed without cotransfecting a promiscuous Gα15 suggested an involvement of Gαq. Interestingly, in contrast to that observed in hBRIE 380i cells, the increased [Ca2+]i in STC-1 cells was not significantly inhibited by a 5-h pretreatment with 50 to 200 ng/ml PTX, an inhibitor of Gαi (data not shown).
GPR93 activation by peptone in STC-1 cells increases CCK transcription.
A signature of STC-1 cells is the production and secretion of CCK. Peptone induces CCK message levels through increased [Ca2+]i (18). Therefore, we asked whether the activation of overexpressed GPR93 generated enough calcium to affect the level of CCK transcript. STC-1 cells overexpressing GPR93 that were treated with 50 mg/ml peptone for 6 h showed an elevated CCK mRNA level compared with vector-transfected cells (2.56 vs. 1.7 fold; Fig. 3). Similarly, 10 μM LPA also stimulated the CCK message level (2.2-fold in GPR93-overexpressing STC-1 cells vs. 1.32 fold in vector-transfected cells). We also tested the message level of GIP in the transfected STC-1 cells. GIP mRNA levels in both the GPR93-overexpressing cells and empty vector transfectants were not affected by peptone or LPA treatments (Fig. 3). This is in agreement with what was observed by others that peptone did not affect GIP transcript level in STC-1 cells (19) and in the intestine in vivo (50).
To determine whether the CCK transcript change produced by peptone in GPR93-overexpressing cells could be caused by a modulation in the CCK transcription level, an 820-bp DNA fragment containing the rat CCK promoter [from 728 bases upstream to 91 bases downstream of the transcription start site; as described by Bernard et al. (2)] was isolated and used to drive the expression of a luciferase reporter gene. A 6-h treatment with peptone increased the luciferase activity in GPR93-overexpressing cells more than that in control cells in a dose-responsive manner (Fig. 4). Surprisingly, we did not observe an induction of reporter activity by LPA up to 10 μM. In the [Ca2+]i mobilization assay, stimulation of [Ca2+]i flux by LPA at concentrations above 1 μM was already at the plateau phase. This indicates that peptone and LPA activation of GPR93 lead to an increase in CCK message level through different pathways. One possibility is that LPA activation of GPR93 could also induce CCK transcription but the DNA element(s) mediating LPA effects was outside the CCK promoter fragment that was used.
It was reported that the calcium-dependent activation of ERK1/2 is involved in the induction of CCK transcription in response to peptone in STC-1 cells (18). Whether ERK1/2 phosphorylation induced by GPR93 activation (10) could be involved in the induction of CCK transcription was determined by using a MEK1/2 inhibitor, PD98059. A treatment with PD98059 at a concentration of 15, 30, 50, or 80 μM caused a dose-dependent decrease in the induction of luciferase reporter activity by GPR93 activation (Fig. 5, solid bars 3 to 6 vs. 2).
A DNA element mediating peptone responses (PepRE) was reported to be overlapping with a cAMP-responsive element/TPA response element at position 72 to 83 bases upstream of the CCK transcription start site (2). Five or 10 μM H-89, an inhibitor of protein kinase A, caused a significant reduction of the increased reporter activity due to GPR93 activation (Fig. 5, solid bars 8 and 9 vs. 2). Similarly, the inhibitor of CaMK, KN93, at a concentration of 3 or 10 μM, also decreased the level of induction of luciferase activity by GPR93 activation in a dose-dependent manner (Fig. 5, solid bars 11 and 12 vs. 2). To verify that the increased [Ca2+]i due to GPR93 activation by peptone was responsible for the induction of the luciferase reporter activity, we used intracellular Ca2+ chelator BAPTA-AM. At a concentration of 20 μM, BAPTA-AM inhibited peptone-induced CCK promoter activity by ∼80% (data not shown). The results of the inhibitor treatments were similar to what has been reported for the endogenous peptone response in STC-1 cells (18).
GPR93 activation enhances CCK secretion of STC-1 cells.
We further explored the effects of peptone in STC-1 cells overexpressing GPR93 by examining the levels of immunoreactive CCK octapeptide (CCK-8) that was released into the media.
We first determined that the basal level of CCK-8 in STC-1 cells was 1.94 ± 0.52 pmol/well (n = 8) and the amount of CCK-8 that was secreted into the culture media in 2 h was 0.04 ± 0.01 pmol/well (n = 8) (2% of the total CCK-8 level). These levels are comparable to what was reported by others (46), indicating that our experimental conditions and methods were adequately sensitive for CCK-8 quantitation.
CCK-8 release in STC-1 cells was significantly induced by 5 and 25 mg/ml (corresponding to 0.5 and 2.5% wt/vol) peptone in a dose and time-dependent manner (Fig. 6, open bars 3, 4, 6, and 7 vs. 1). Consistent with the induction of CCK transcription described above, CCK-8 secretion was also enhanced in response to peptone in GPR93-overexpressing STC-1 cells compared with that in the control cells (Fig. 6, solid vs. open bar 6 and 7). Interestingly, similar to the CCK promoter-reporter activity, CCK-8 release was not significantly induced by 1 μM LPA (Fig. 6, bars 8 to 10 vs. 1) even though 1 μM LPA and 25 mg/ml peptone increased the [Ca2+]i in GPR93 transfected STC-1 cells to a similar extent. This suggests that activation of GPR93 by LPA alone is not enough to induce the release of CCK-8 in STC-1 cells.
GPR93 activation by peptone and LPA increases intracellular cAMP concentration.
Since H-89 significantly inhibited the peptone-induced CCK promoter reporter activity, we sought to determine whether intracellular cAMP was involved. Additionally, GPR93 has been reported to increase intracellular cAMP in response to LPA in neural cells (29) and gastrointestinal lymphocytes (28). Therefore, we tested whether peptone- or LPA-induced GPR93 activation can lead the elevation of intracellular cAMP in STC-1 cells. A treatment with 50 mg/ml of peptone for 7 min increased the intracellular cAMP in STC-1 cells by 8-fold (Fig. 7, open bar 2 vs. 1), which was further enhanced up to 16-fold in GPR93 STC-1 cell transfectants (Fig. 7, solid bar 2 vs. 1). LPA, at 10 μM, only increased the intracellular cAMP by 1.5- and 2-fold in vector- and GPR93-transfected STC-1 cells, respectively (Fig. 7. bar 3 vs. 1). The differential effect of LPA and peptone activated GPR93 on intracellular cAMP might be reflected on the responsiveness of the CCK transcription and secretion to peptone but not to LPA. This would be consistent with the reported observations that an increase in both intracellular cAMP and calcium is required for the induction of reporter activity linked to the same minimal CCK promoter (18), and elevated intracellular cAMP induces the release of CCK in STC-1 cells (8).
The results of this study indicate that the overexpressed GPR93 in STC-1 cells can be activated by peptone and by LPA. Although the observed increase in [Ca2+]i in response to peptone and LPA is of a similar magnitude, peptone and LPA seem to have different effects on the regulation of intracellular cAMP level. The overexpression of GPR93 in STC-1 cells magnified several of the effects of peptone observed by others such as the modulation of the levels of CCK transcript, CCK transcription, and CCK-8 secretion. The present study brings forth the possibility of a GPCR(s) acting as an enteroendocrine cell transducer of extracellular stimuli for bioactive peptide secretion.
CCK is physiologically important for the stimulation of pancreatic enzyme secretion, inhibition of gastric emptying, enhancement of amino acid-stimulated insulin secretion, stimulation of intestinal motility, and inhibition of food intake (see Ref. 34 for review). Dietary fat and protein in the lumen of the intestine are the major physiological stimuli for CCK secretion. Intact proteins can stimulate the release of CCK by inhibiting the trypsin digestion of the CCK-releasing peptides that are from the pancreatic juice (i.e., monitor peptide) (26) and the intestinal lumen (which is identical to porcine diazepam binding inhibitor) (24, 47). Digested proteins (20, 27) or enzymatic hydrolysate of proteins have also been demonstrated to be strong stimulants of CCK release (15). The presence of those luminal nutrients clearly has to be “sensed” by the intestinal CCK releasing I cells. However, the sensing mechanism that leads to the induction of CCK release is still not clearly defined since the activation of CCK release is a highly integrative event involving neuronal, hormonal, and luminal factors, which can be mediated by direct or indirect effectors.
The possibility that a GPCR in enteroendocrine cells is involved in the luminal sensing has been partly suggested by the identification of the bitter taste receptor, T2R, in enteroendocrine cells (43). In STC-1 cells that endogenously express T2R, gustatory Gαi proteins gustducin, and transducin, denatonium benzoate (DB) and phenylthiocarbamide (PTC) increase [Ca2+]i through the opening of the l-type voltage-gated calcium channel (VGCC), which parallels the release of CCK (9). In oral neuroendocrine cells, DB and PTC also induce a rise in [Ca2+]i due to an influx of extracellular calcium (presumably through T2R activation) and not due to mobilization from intracellular stores (9, 23, 35).
Besides stimulating gastrointestinal bioactive peptide release, luminal nutrients are also capable of inducing the expression of those peptide genes. For example, transcription of proglucagon (19) and CCK (13) genes are also induced by dietary protein hydrolysate. The processes involved in the changes in CCK gene expression in response to luminal stimuli such as protein hydrolysate are still not completely defined. Although indirect effects of luminal stimuli on the secretion and synthesis of CCK in I cells are likely, such as through paracrine intermediates or vagal or myenteric nerves (6, 16), there is evidence for a direct effect of protein hydrolysate on CCK mRNA levels in STC-1 cells (18), which appears to be by an intestinal-specific nutrient-dependent mechanism in enteroendocrine cells (13).
One of the cellular factors that has been proposed to mediate protein hydrolysate stimulation of CCK release is the proton-coupled oligopeptide transporter PepT1 (36). However, STC-1 cells do not express PepT1 (36, 39), and yet protein hydrolysate still stimulates CCK secretion and transcription (13).
A peptone-induced CCK release in STC-1 cells has been shown to occur through a partially PTX-sensitive pathway (39), indicating a GPCR(s) involvement in this process. However, peptone only moderately induces an accumulation of intracellular second messengers compared with bombesin, and peptone or cephalexin induction of CCK secretion is not sensitive to PKA or PKC inhibitors (39). These data suggest that a GPCR might not be involved in the release of CCK (39). PTX-sensitive peptone or cephalexin-induced CCK secretion could be a result of [Ca2+]i influx through VGCCs [such as L-type channels acting via PTX-sensitive G proteins (21, 22, 25, 39, 42)] that consequently stimulates CCK release (39) since VGCC blockers such as diltiazem reduce [Ca2+]i, which, results in decreased CCK secretion (9).
GPR93, which was initially identified in a number of tissues (30), is highly expressed in the small intestinal mucosa (10). GPR93, also known as GPR92, was reported to bind LPA (28, 29), and its activation leads to Gαq-, Gαs-, Gαi-, and Gα12/13-mediated pathways (10, 28, 29). In response to protein hydrolysate alone or synergistically with LPA, the activation of GPR93 in the nontumorigenic hBRIE 380i enterocyte cell line induces intracellular calcium levels. In contrast to the T2R activation in response to DB or PTC, the activation of GPR93 by peptone or LPA in enterocytes increases [Ca2+]i that is independent of the influx of extracellular calcium and can be blocked with 20 nM thapsigargin (an endoplasmic Ca2+-ATPase inhibitor) as well as the PLCβ inhibitor U-73122 (10). Therefore, the activation of GPR93 by protein hydrolysate may not lead to downstream responses that are dependent on the influx of extracellular calcium. The identification of a GPCR in the enterocytes that is directly responsive to protein hydrolysate, resulting in increased intracellular calcium level when activated, opened the possibility that the activation of this receptor in enteroendocrine cells might be part of the sensing mechanism responsible for the release and synthesis of CCK in response to protein hydrolysate.
We determined that GPR93 is expressed in STC-1 cells. In the GPR93-overexpressed STC-1 cells, peptone and LPA induced [Ca2+]i in a dose-dependent manner. In agreement with other studies (2, 13, 18), we also observed the induction of CCK message level by protein hydrolysate in STC-1 cells. The activation of GPR93 by protein hydrolysate and LPA enhanced the increased CCK message level. However, only peptone increased the luciferase reporter activity driven by a fragment of CCK promoter containing the cis-acting element (PepRE) that confers peptone regulation of CCK transcription. Despite the inherent responsiveness of STC-1 cells to protein hydrolysate, STC-1 cells constitutively expressing GPR93 exhibited a significant increase in CCK promoter-driven reporter activity when this receptor was activated. Using a intracellular calcium chelator, BAPTA-AM, and chemical inhibitors, we determined that GPR93 induction of CCK promoter activity depended on elevated [Ca2+]i through the activation of ERK1/2-, CaMK-, and PKA-mediated pathways, which is consistent with what was reported by others (18). Our observation that both LPA and peptone induced the CCK transcript but only peptone increased reporter activity from a CCK minimal promoter suggests that LPA-responsive elements might be upstream of the DNA element responsive to peptone. Another possibility is that peptone and LPA might affect CCK message stability differently.
A rise in [Ca2+]i is necessary for CCK secretion. Activation of the bombesin receptor induces exocytosis of CCK by increasing [Ca2+]i (46). Activation of β1- or β2-adrenergic receptors in STC-1 cells results in the Ca2+-dependent release of CCK through a process coupled to cAMP production and the influx of extracellular calcium through calcium channels (44). However, peptone and LPA might have different effects on CCK processing and/or secretion since we only observed an induction of CCK release by peptone. This could be due to the higher induction of intracellular cAMP by peptone compared with LPA in GPR93-overexpressing STC-1 cells. Besides increased level of [Ca2+]i, intracellular cAMP is another well-studied regulator of secretory granule exocytosis in many cells types including pancreatic β-cells and enteroendocrine cells (45). Treatments of STC-1 cells with agents that give rise to cAMP, such as forskolin, or cAMP analogs, stimulate CCK release (8). GLP-1, a hormone released from epithelial cells of the gastrointestinal mucosa that enhances the glucose-mediated insulin secretion in pancreatic β-cells, activates its specific receptor, resulting in an increase of both intracellular cAMP and calcium (49). In RINm5F cells, subcellular targeting of PKA through association with A-kinase-anchoring proteins is needed to facilitate cAMP-mediated elevation of intracellular calcium and GLP-1 mediated insulin secretion (32). Additionally, A-kinase-anchoring proteins themselves can modulate GPCR activation and also act as downstream effectors (1), therefore providing another point of regulation that may be sensitive to intracellular cAMP concentration. Intracellular cAMP could also affect the release of CCK by altering the pool size of release-ready secretory vesicles via PKA [similar to what occurs in chromaffin cells from bovine adrenal glands (38)].
The activation of GPR93 by its activators, peptone and LPA, resulting in differential effects on CCK release might be due to coupling to different G proteins. Examples of this variability in coupling due to specific agonists activation have been observed for a number of GPCRs. The adenosine receptor A1 leads to differential activation of Gαi, Gαs, and Gαq due to specific conformational changes as a result of small differences in the ligand structure (12). Activation of calcium-sensing receptor by calcium and l-phenylalanine produces sinusoidal and transient [Ca2+]i oscillations, respectively, mediated by different pathways (41). The efficacies of six N-formyl peptide agonists of the N-formyl peptide receptor in human neutrophils are of the same kind but with a range of potencies spanning three orders of magnitude (48). In pituitary cells, activation of endothelin (ET) receptor, which has differing affinity for ET-1, ET-2, and ET-3, is coupled to multiple G proteins (Gαs, Gαq, and Gαi/o), resulting in the regulation (excitation or inhibition) of prolactin secretion (3). The ET pathways are mediated by Gα as well as Gβγ affecting [Ca2+]i and intracellular cAMP levels, the plasma membrane potassium and calcium channels, and the exocytotic machinery (3). It appears that GPR93 could be yet another example of a GPCR whose G protein coupling is dependent on the species of the agonist.
In the present study, we have demonstrated a new mechanism of protein hydrolysate regulation of CCK gene expression and secretion through direct activation of a GPCR, GPR93. Physiologically, it may be the functional integration of this activation along with other cellular systems that form the luminal sensory system. This luminally responsive system could include oligopeptide transporters such as the PepT1 (16), G protein-dependent and -independent activation of VGCC inducing influx of external calcium, and other indirect regulators of the CCK secretion such as L-type VGCC and CCK-releasing peptides. Additionally, paracrine action by other cells of the mucosa that could be responsive to luminal stimuli, such as nerves of the myenteric plexus and enterochromaffin cells, adds another layer of regulation to this possible sensory system. Although it remains to be determined whether GPR93 initiates the same responses in enteroendocrine cells in situ as observed in STC-1 cells, the present study demonstrates the potential importance of GPCRs as transducing molecules of luminal dietary signals.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-58592 (to G. W. Aponte).
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